The genomics revolution has repeatedly demonstrated that our understanding of natural product (NP) biosynthesis is far from complete. Given the frequency of silent and cryptic biosynthetic clusters in existing and emerging genomes, compounded with the inability to culture >99% of microbes, far less than 1% of microbial NPs have been discovered. This project proposes to characterize novel NP biosynthetic clusters from two soil-dwelling bacteria. Without question, NPs from soil bacteria are our most prolific source of medicine. Unlocking the chemical structure and biological function of novel NPs encoded by these organisms holds enormous potential for expanding our pharmaceutical repertoire. The biosynthetic clusters of interest to this proposal are members of a recently described, evolutionarily conserved family dubbed the thiazole/oxazole-modified microcins (TOMM). As a new NP family, TOMMs represent an underexplored area of NP chemical space - few have a known structure or mechanism of action. All TOMMs with a known activity function as toxins, making them of paramount interest to modern medicine. In two known cases, TOMMs produced by human pathogens play a critical role in the molecular mechanism of pathogenesis. Therefore, a more complete knowledge of the biosynthetic pathway could lead to the development of virulence-targeting antibiotics, which represents a longer-term objective for our research program. To effectively tap into this potential, several gaps in our current understanding of these molecules must be addressed. This project is divided into three related, but independent specific aims. For Aim 1, a combination of in vitro reconstitution, natural product isolation, and advanced spectroscopy will be employed to determine the chemical structure of the TOMM product. In Aim 2, high-resolution mass spectrometry and site-directed mutagenesis will be used to kinetically evaluate a key enzyme that catalyzes the first step in the formation of thiazoles and oxazoles. This enzyme, a cyclodehydratase, is responsible for recognizing the TOMM precursor peptide and converting Cys and Ser/Thr residues into thiazolines and (methyl) oxazolines. Aim 3 seeks to reveal the protein-protein interactions that enable substrate recognition and the downstream thiazole/oxazole forming activity. By characterizing the enzymes involved in TOMM biosynthesis, the foundation for future work will be laid, including the development of biosynthetic inhibitors of TOMMs from human pathogens and strategies to harness the power of combinatorial biosynthesis to evolve TOMMs with desired biological targets. Progress on this project will fill a major void in our current understanding of how a subset of peptide-derived toxins is biosynthesized. The tools developed will be broadly applicable to the study of other TOMMs.